Serine MARylation (Ser-MARylation), catalyzed by poly(ADP-ribose) polymerase 1/2 (PARP1/2)-Histone PARylation factor 1 (HPF1) complex and reversed by ADP-ribosyl-acceptor hydrolase 3 (ARH3), is the primary form of DNA damage-induced ADP-ribosylation in human cells. Distinct from PARylation, Ser-MARylation represents a critical "second wave" of signaling following DNA damage. Dysregulation of Ser-MARylation is implicated in neurodegeneration and cancer, and ARH3 deficiency drives resistance to PARP inhibitor therapy. However, mechanistic characterization of Ser-MARylation reversal has been hampered by the lack of well-defined protein substrates and sensitive quantitative tools. Here, we report a quantitative assay to monitor ARH3-mediated reversal of protein-linked Ser-MARylation using Ser-MARylated HPF1 as a specific, defined substrate. Strikingly, ARH3 displays a greater than 200-fold kinetic preference for Ser-MARylated HPF1 over MARylated PARP1. By using a panel of ADP-ribose analogs, we identified ADP-HPD as an effective ARH3 inhibitor that mimics the oxocarbenium-like transition state of O-glycosidic bond hydrolysis. Together with structural and mutational analysis, these findings support a catalytic mechanism in which Asp77 activates a catalytic water for nucleophilic attack on the scissile serine-ADP-ribose bond. Collectively, this work establishes a mechanistic framework for Ser-MARylation reversal and lays the groundwork for rational ARH3 inhibitor development.

Drug resistance remains a major limitation to effective cancer therapy, and its metabolic basis is incompletely understood. We recently developed models of acquired drug resistance through chronic exposure of tumor cells to anticancer agents and identified a metabolic phenotype characterized by impaired mitochondrial bioenergetics, increased L-lactate production, and decreased oxygen consumption rate (OCR). Here, we investigated the contribution of D-lactate metabolism to this phenotype. Compared with their parental (P) counterparts, drug-resistant (R) cell lines showed increased D-lactate production together with increased glyoxalase 1 (GLO1) expression and activity. Sodium butyrate (NaB) reduced GLO1 expression and activity in tumor cell lines with higher expression of D-lactate pathway components, and NaB treatment was associated with reduced intracellular D-lactate levels and increased sensitivity to the corresponding anticancer treatments in selected cell models. These findings support a biochemical link between NaB-mediated modulation of the GLO1/D-lactate pathway and drug response, without excluding additional NaB-dependent mechanisms. Furthermore, NaB enhanced the effects of pro-differentiation agents in the tested in vitro models. Together, our data identify the GLO1/D-lactate pathway as a metabolic feature associated with drug resistance and support further investigation of NaB-based strategies in appropriate preclinical models.

Polyphosphate (polyP), a linear polymer of orthophosphate residues, is enriched in secretory granules in specialised mammalian cell types including platelets and mast cells. Although polyP released during activation and degranulation of these cells has been shown to promote blood clotting and inflammation, little is known about the mechanisms governing polyP synthesis in these granules. In mice, the loss of IP6K1, an enzyme that catalyses the production of 5-InsP7, has been shown to result in depletion of platelet polyP and impaired hemostasis. Here, we use the rat mast cell line RBL-2H3 as a model to study the regulation of polyP synthesis in secretory granules. By monitoring real-time polyP synthesis in isolated mast cell granules, we demonstrate that ATP is the substrate fuelling granule polyP production. By the use of inhibitors, we show that accumulation of polyP in granules requires an intact transmembrane proton gradient maintained by vacuolar H+ATPase (V-ATPase). In RBL-2H3 cells, depletion of IP6K1 led to a substantial reduction in cellular polyP levels and defective accumulation of polyP, serotonin, and tryptase inside granules. Cells with reduced IP6K1 showed a profound loss of granule acidification, correlating with downregulated levels of V1 subunits of V-ATPase. Adding back active or catalytically inactive IP6K1 rescued the expression of V-ATPase V1 subunits, reversed granule deacidification, and restored polyP levels in IP6K1-depleted cells. Together, these data unveil a role for IP6K1 in maintaining granule pH and thereby supporting polyP synthesis in mammals.

Mechanosensitive (MS) channels are essential transducers that convert mechanical stimuli into biochemical signals. OSCA/TMEM63 proteins constitute a conserved family that serves as mechanical and osmotic stress sensors, yet the mechanisms by which the chemical microenvironment modulates their gating remain unclear. Here, we identify intracellular pH as a potent and side-specific regulator of OSCA/TMEM63 activity. Using patch-clamp electrophysiology, we demonstrate that OSCA/TMEM63 currents are reversibly tuned by pH: intracellular acidification suppresses channel activity, whereas alkalinization markedly potentiates it. This regulation is mediated by a pronounced shift in the mechanical activation threshold, thereby effectively tuning channel sensitivity. Our findings establish intracellular pH as a conserved modulator of OSCA/TMEM63 mechanosensitivity, providing a mechanistic framework for understanding how the intracellular chemical milieu is coupled to osmo/mechano sensing.

Macrophages are activated by infection via TLR signaling and produce pro-inflammatory cytokines, including TNF. However, enhancers regulating TNF transcription in human macrophages have not been well investigated. Here, using next-generation sequencing methods, we identify six candidate enhancers within a 131 kb region around the TNF locus in primary human alveolar macrophages. Three enhancers -distal -16kbTNF, -7.1kbTNF, and proximal enhancer PETNF -directly regulate TNF transcription upon TLR3 stimulation. Deletion of the distal -16kbTNF enhancer led to a 73% reduction in TNF gene transcription in response to poly(I:C) stimulation. Additionally, deletion of the -7.1kbTNF enhancer resulted in a 41% reduction in TNF mRNA, while deletion of the PETNF enhancer resulted in a 52% reduction. A high-throughput reporter assay identified DNA fragments that drive enhancer activity. Mutagenesis analysis of transcription factor binding motifs revealed that PU.1-binding sites are required for enhancer activity. Further analysis demonstrates that PU.1, rather than EGR1 or AP-1, is the master regulator, regulating TNF enhancer activities and TNF transcription. This study shows that PU.1 plays a critical role in driving TNF enhancer activity and mRNA expression in human macrophages.

A hallmark of Schitoviridae bacteriophages is a giant (∼3,500-residue) virion-associated RNA polymerase (vRNAP) that is ejected into the host to initiate early transcription. Structural and functional characterization of vRNAP has thus far been limited to coliphage N4, leaving the conservation of vRNAP architecture, promoter recognition, and regulation across Schitoviridae largely unexplored. Here, we characterized the vRNAP from the schitovirus DEV, a lytic phage of Pseudomonas aeruginosa used in experimental phage therapy cocktails. Cryo-EM structures of DEV vRNAP (gp71) in the apo state and bound to its cognate ssDNA hairpin promoter reveal that DEV vRNAP adopts a conserved closed-fist, single-subunit RNA polymerase fold and recognizes a partially folded ssDNA hairpin promoter, similar to phage N4 vRNAP, despite sharing only 20% sequence identity. The ∼140-kDa C-terminal domain (CTD) adopts a trapezoidal, predominantly α-helical architecture comprising two subdomains with only weak structural similarity to the N4 CTD (RMSD ∼15.3 Å). The RNAP domain and CTD are connected by a short linker but remain highly dynamic, forming a compact assembly in only a small fraction of particles, whereas the N-terminal domain, which has proposed membrane-binding activity, was not resolved. Radiolabeled runoff transcription assays demonstrated that the CTD strongly inhibits transcription both intramolecularly, in cis, and as an isolated domain, in trans. Together, these findings reveal that DEV and N4 vRNAPs conserve a common RNAP fold, promoter-recognition mechanism, and CTD-mediated autoinhibition despite substantial sequence and structural divergence, defining conserved principles of Schitoviridae vRNAP architecture and function.

AFG3L2, located in the mitochondrial inner membrane, is an ATP-fueled protease responsible for protein degradation, and its dysfunction has been implicated in multiple neurodegenerative disorders. However, the structural basis underlying its ATP-driven substrate translocation remains incompletely understood. Here, we purified full-length human AFG3L2 and determined its cryo-EM structures in four distinct conformational states (Con1-Con4), revealing substrate engagement in the core catalytic domain despite weak EM density for its transmembrane domain. These structures display an asymmetric spiral-staircase architecture in which the six protomers are arranged around a central pore that accommodates a polypeptide substrate. The four conformational states jointly reveal a substrate translocation mechanism in which ATP binding by the uppermost protomer promotes substrate engagement, whereas ATP hydrolysis by the lowermost ATP-bound protomer leads to substrate release. Our findings provide snapshots of ATP-driven substrate translocation by AFG3L2 and refine the hand-over-hand translocation mechanism by revealing substrate capture and release during an ordered ATPase cycle.

Humanin (HN), a mitochondria-derived peptide, exerts cytoprotective effects by modulating apoptosis through interactions with pro-apoptotic BCL-2 proteins, BAX and BID. Previous studies showed that HN induces β-sheet-rich fibrilization of BAX and truncated BID, suggesting a mechanism that may explain its cytoprotective function. Mutants of HN were shown to alter the morphology and fibrilization with BAX. In this study we tested whether the same HN variants can also influence fibrilization with BID, cleaved BID, or active truncated BID. We also evaluated the impact of fibrils formed between BAX or BID with HN on membrane morphology and permeability. The single Cysteine, C8, in HN is critical for fibrilization with all forms of BID. Whereas the central hydrophobic residues, L9 and L10, modulate fibril morphology with BID and cleaved BID; as they did with BAX. Interestingly, L12 has a differential effect on fibrilization with BID and BAX. Substitutions to the above residues of HN prevent formation of fibrils with truncated Bid. The HN fibril promotes vesicles to assemble as shown by electron microscopy, and this is moderated in the presence of BAX or BID. Vesicle leakage assays further demonstrated that HN reduced BAX- and tBID-mediated membrane permeabilization. Importantly, inhibition was strongly dependent on the order of sample addition. Finally, HN fibrils densely packed on isolated mitochondria in an organized parallel configuration. These findings extend the model of HN action by integrating BID activation-state specificity, HN residue-level determinants, and direct membrane association into a unified framework for HN-mediated regulation of mitochondrial apoptosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver disorder, with its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), characterized by steatosis, inflammation, hepatocyte injury, and fibrosis. However, the underlying mechanism remains incompletely understood. Here, we performed an in vivo CRISPR screen in Cas9 mice fed a methionine- and choline-deficient (MCD) diet to identify epigenetic regulators of MASH. SETDB1 emerged as a key modulator: hepatocyte-specific loss of Setdb1 exacerbated steatosis, inflammation, ductular reaction, and fibrosis, whereas Setdb1 overexpression markedly alleviated these pathological features. Notably, human liver samples showed decreased SETDB1 expression with advancing MASLD. Furthermore, SETDB1 deficiency increased hepatic ferroptosis by upregulating ACSL4, aggravating MASH pathology, whereas ACSL4 knockout reduced liver injury in the absence of SETDB1. SETDB1 overexpression protected against liver damage by repressing ACSL4. Therefore, the SETDB1-ACSL4-ferroptosis axis regulates MASH progression and represents a potential therapeutic target.

Intramuscular fat, deposited as marbling in beef, is a key economic indicator of meat quality in cattle production. A series of circular RNAs (circRNAs) have emerged as critical regulators of lipogenesis. In this study, we identified 373 differentially expressed circRNAs between calves and adult cattle, among which circINSR was markedly upregulated in adults. Single-nucleus RNA sequencing (snRNA-seq) analysis revealed the developmental trajectory of intramuscular adipocytes, demonstrating the differentiation of preadipocytes into both white and beige adipocytes. Along the preadipocyte-to-white adipocyte differentiation axis, 133 genes were significantly upregulated in white adipocytes, with notable enrichment in the INSR-mediated PI3K-Akt signaling pathway. Functional studies in vitro and in vivo confirmed that circINSR promotes adipocyte differentiation, facilitates fatty acid transport and synthesis, enhances triglyceride accumulation, and drives lipid droplet maturation through PI3K-Akt signaling. Mechanistically, circINSR functions as a competitive endogenous RNA (ceRNA) by sequestering miR-712-5p, thereby upregulating INSR expression and potentiating lipogenesis. The results delineates the cellular heterogeneity and differentiation program of intramuscular adipocytes in bovine longissimus dorsi muscle and establishes circINSR as a miR-712-5p sponge that orchestrates lipogenesis. The findings advance our understanding of the molecular mechanisms governing IMF deposition in high-quality beef cattle.

Bacteroides are abundant gut bacteria with diverse polysaccharide metabolizing capabilities encoded by co-transcribed polysaccharide utilization loci (PULs). The starch utilization system (Sus) is required for starch and α-glucan metabolism in Bacteroides thetaiotaomicron and has served as a model for PUL studies. However, the mechanism of maltooligosaccharide (MOS) transport and the architecture of the SusCD complex remain incompletely understood. Here, we used cryo-electron microscopy to capture multiple transport-relevant conformations of the SusD lipoprotein and the TonB-dependent transporter SusC in both unliganded and ligand-bound states. In the absence of ligand, SusCD adopts three conformations including an open state in which SusD is displaced from SusC, a state containing the SusC plug, and a state lacking density for the SusC plug. Three-dimensional variability analysis of the structure where SusD is displaced from SusC reveals continuous SusD mobility coupled to movement of the SusC N-terminus. The maltoheptaose-bound SusCD structure reveals two binding sites, one at the SusD-SusC interface and a second within SusC, indicating how MOS stabilizes a closed SusCD assembly. Notably, all structures show that SusCD is a heterodimer, unlike other Sus-like transporters that assemble into SusCD heterotetramers. Native mass spectrometry confirms this heterodimeric stoichiometry, and comparisons with other SusCD-like complexes suggest a structural basis for this distinct assembly of SusCD. Together, these data define substrate binding and intermediate transport states in a canonical PUL system and reveal architectural divergence among Sus-like carbohydrate transport complexes in Bacteroides.

Compound heterozygous cells have two different disease-causing variants on different alleles of the same gene. Targeting one of the variants in a compound heterozygous cell with Cas9 nuclease or D10A nickase, plus an allele-specific guide RNA that is specific for one of the mutations, has been shown to trigger allele conversion and restore the wild-type sequence to the targeted allele. To gain insights into this process, we designed and created a Compound Heterozygous Allele Conversion Reporter cell line comprising two near-identical alleles with wild-type or mutant versions of three different fluorescent reporter genes. The cell line was designed with mutations that prevent expression of a functional mCherry reporter protein, but create protospacer adjacent motifs for Cas9 recognition which can be selectively targeted with allele-specific gRNAs. When transfected with either Cas9 nuclease, D10A nickase or ABE8e, plus an allele-specific gRNA targeting the allele containing the mCherry[fs-2,Y77X] variant, >10% of cells recover mCherry fluorescence. Sorting and sequencing these mCherry-fluorescent cells revealed wild-type mCherry sequence indicative of allele conversion of the mCherry[fs-2,Y77X] variant using the homologous allele as a repair template. Allele conversion efficiency is increased by inhibiting DNA-PKcs but is reduced by overexpression of dnMLH1 or Rad51.

Transcriptional regulation by coregulators and other proteins is essential for osteoblast differentiation. Bone density maintenance requires tissue-specific androgen and androgen receptor (AR)-regulated signaling in osteoblasts. However, the transcriptional mechanisms through which androgens exert specific effects on osteoblasts remain unclear. Here, to explore androgen signaling in osteoblastic cells, we investigated comprehensive AR-binding sites by using chromatin immunoprecipitation and sequencing (ChIP-seq) analysis. We observed that the transcription factor (TF) Fra-1, an important player for osteoblast physiology, modulates epigenetic states to determine the AR cistrome and osteoblastic cell differentiation. The binding of Fra-1 to specific genomic regions in human osteoblastic cells facilitates the binding of AR to regulatory sequences, leading to active transcription. We demonstrated that the super-enhancers encoding critical developmental factors in osteoblastic cells overlapped with osteoblastic cell-specific AR-binding sites. Of note, overexpression of Fra-1 in neuronal cells, which are derived from other tissues with low baseline Fra-1 expression, we observed that Fra-1 overexpression facilitates the formation of super-enhancers associated with osteoblastic features, including the RUNX2 and TGF-β signaling pathways, by altering comprehensive histone modifications and the AR cistrome. Thus, we propose that interaction with critical transcription factors for osteoblastic cell function can govern the androgen response and epigenetic regulation involved in bone differentiation. The present findings significantly advance our understanding of sex steroid-mediated regulation of bone homeostasis.

Cold exposure induces adaptive molecular and mitochondrial remodeling in brown adipose tissue (BAT) of mice to sustain thermogenesis. We previously showed that the lipid droplet protein Perilipin 5 (PLIN5) is strongly induced in BAT by cold exposure and promotes thermogenic gene expression, mitochondrial remodeling, and uncoupled respiration. However, constitutive BAT-specific PLIN5 deletion impaired mitochondrial function without causing cold intolerance, suggesting physiological compensation, likely via induction of thermogenic genes in white adipose tissue (WAT). To test this possibility, we generated mice with doxycycline-inducible BAT-specific PLIN5 deletion. Acute PLIN5 deletion in adult mice during cold exposure reduced BAT thermogenic gene expression, mitochondrial cristae density, and uncoupled mitochondrial respiration, which resulted in cold intolerance. Unlike constitutive PLIN5 knockout mice, mice acutely rendered deficient of PLIN5 in BAT failed to mount a compensatory thermogenic response in inguinal WAT. Together, these findings demonstrate that PLIN5 is required for mitochondrial homeostasis in brown adipocytes and for adaptive thermogenesis during cold exposure. Notably, by cell fractionation and protease protection assay, we observed that PLIN5 enriches on the outer mitochondrial membrane in BAT during cold exposure. We also observed that the absence of PLIN5 in BAT is not associated with reduced contacts between lipid droplets and mitochondria, thereby suggesting that the role of PLIN5 in adaptive thermogenesis may depend on its translocation to the mitochondrial outer membrane for a purpose unrelated to the formation of contacts between lipid droplets and mitochondria.

FAT1 is a well-established tumor suppressor in gastric cancer (GC), yet how its loss drives chromosomal instability remains poorly understood. Although FAT1 has been implicated in homologous recombination through Hippo signaling, it is unknown whether FAT1 directly participates in the DNA damage response (DDR) within the nucleus. Here, we show that a FAT1 C-terminal fragment (FAT1[CTF]) translocates to the nucleus and nuclear FAT1[CTF] broadly suppresses double-strand break repair by impairing both non-homologous end joining (NHEJ) and homologous recombination (HR). Through TurboID-mediated proximity labelling, we identified the histone acetyltransferase TIP60 as a critical nuclear interactor of FAT1[CTF]. Mechanistically, FAT1[CTF] binds to and inhibits the acetyltransferase activity of TIP60, thereby attenuating the DDR. Clinically, the expression of FAT1 and its sheddase ADAM10 is negatively correlated with DNA repair capacity in GC patients. Collectively, our study defines a protease-dependent 'ADAM10-FAT1[CTF]-TIP60' axis through which FAT1 processing actively suppresses DNA repair, providing a mechanistic basis for its tumor-suppressive function and a potential target to overcome chemoresistance in GC.